Cloning vectors and pBR322
A cloning vector carries foreign DNA into a host. It needs an ori, a selectable marker and single cloning sites; pBR322 is a classic E. coli vector.
What a vector does
A cloning vector is a DNA molecule that carries a foreign gene into a host cell and replicates there. Plasmids (small, circular DNA outside the bacterial chromosome) and bacteriophages are used because they replicate inside bacteria independently of the chromosome. If a foreign DNA is linked to them, it is copied as many times as the vector: its copy number. Modern vectors are engineered so that foreign DNA is easy to link and recombinants are easy to select.
Features a cloning vector needs
| Feature | What it does |
|---|---|
| Origin of replication (ori) | Replication starts here, so any DNA linked to it replicates in the host. It also controls the copy number: to get many copies, use a vector whose ori supports a high copy number. |
| Selectable marker | Helps identify and eliminate non-transformants and lets only transformants grow. Genes for resistance to ampicillin, chloramphenicol, tetracycline or kanamycin are used, as normal E. coli resists none of these. (Transformation: putting a piece of DNA into a host bacterium.) |
| Cloning sites | Very few, preferably one, recognition site for each commonly used restriction enzyme. More sites would cut the vector into several pieces and complicate cloning. Foreign DNA is usually ligated into a site inside one antibiotic resistance gene. |
| Vectors for plants and animals | A disarmed Ti plasmid of Agrobacterium tumefaciens for plants; disarmed retroviruses for animal cells. |
How to draw pBR322
- Draw a large circle for the double-stranded plasmid and write pBR322 in the centre.
- Mark ori at the bottom and rop just beside it (rop codes for proteins involved in plasmid replication).
- Draw a thick arc on the left labelled ampR; mark Pst I and Pvu I sites on it.
- Draw a thick arc on the right labelled tetR; mark BamH I and Sal I sites on it.
- Mark EcoR I, Cla I and Hind III near the top, between ampR and tetR, and Pvu II on the lower right near rop.
- Write the title: E. coli cloning vector pBR322.
Selecting recombinants with pBR322 (insert placed in the BamH I site of tetR)
| Kind of cell | On ampicillin | On tetracycline |
|---|---|---|
| Not transformed (no plasmid) | Does not grow | Does not grow |
| Non-recombinant (plasmid without insert) | Grows | Grows |
| Recombinant (insert inside tetR) | Grows | Does not grow |
Two ways to spot recombinants
Both work by insertional inactivation: the insert switches off the gene it lands in.
Vectors for plants and animals
Agrobacterium tumefaciens, a pathogen of several dicot plants, delivers a piece of DNA called T-DNA into plant cells. This turns normal cells into a tumour that makes chemicals the bacterium needs. Its Ti (tumour inducing) plasmid has been modified so it no longer causes disease but still delivers genes of interest into many plants. In the same way, retroviruses, which can make animal cells cancerous, have been disarmed and now deliver useful genes into animal cells.
The disarmed Ti plasmid of Agrobacterium is a common way to put new genes into crop plants such as cotton. Without a good vector there would be no transgenic crop and no recombinant medicine.
5 marks: “Draw a labelled diagram of the E. coli cloning vector pBR322. Explain how insertional inactivation helps to select recombinants.” Diagram: ori, rop, ampR, tetR, and the restriction sites. Insert foreign DNA in the BamH I site of tetR; plate on ampicillin (transformants grow), then on tetracycline (recombinants fail to grow, non-recombinants grow). Alternative: insert in β-galactosidase gene; recombinants give colourless colonies, others blue.
3 marks: “List three features required in a cloning vector and give the role of each.” Ori: starts replication, controls copy number. Selectable marker: lets only transformants grow (e.g. ampR). Cloning sites: single recognition sites so one cut opens the vector.
2 marks: “Why should a cloning vector have only one recognition site for the restriction enzyme used?” One cut simply opens the vector to take the insert. Several sites would cut it into many fragments and complicate cloning.
A vector needs O-S-C: Ori to copy, Selectable marker to choose, Cloning site to cut.